Programmed cell death or apoptosis plays a central role in embryogenesis, morphogenesis and several pathological conditions like neurodegenerative diseases, neoplasia and ischemic injury. The mechanisms of apoptosis and its manipulation are areas of intense current investigation. The morphologic, functional and biochemical aspects of apoptosis have been extensively characterized in cell culture systems. This was accomplished with the help of a powerful array of microscopic and biochemical techniques. In contrast, our understanding of the apoptotic process in animal tissues and organs has been less complete and satisfactory. This is a consequence of the necessity of studying tissues processed ex vivo and harvested at fixed points in time. This approach suffers from substantial loss of information on the temporal evolution of the apoptotic process in live animals. Furthermore, the temporo-spatial development of apoptosis in various parts of complex tissues is, difficult to dissect when examined in a single section harvested at a specific time. To circumvent this problem, our proposed pilot study will examine the feasibility of studying the development of apoptosis in organs of live animals using two-photon fluorescence microscopy. The central hypothesis to be tested is that live imaging of apoptosis in animal tissues, with both 3D and time resolution, is a superior alternative to traditional ex vivo techniques. We will specifically investigate the possibility of systemic labeling of the kidneys in a live animal with fluorescent probes commonly utilized for the study of apoptosis. Criteria for normalcy, apoptosis and necrosis will be established based on correlative studies with ex vivo or fixed tissues. We also propose to study the live evolution of apoptosis over time and space in a determined volume of kidney tissue following ischemic injury. These studies have the potential to yield substantial and new information on the apoptotic process. They might also uncover hitherto unsuspected aspects of this complex and important mode of cell death.